Methods of enzymatic fecal bile acid analysis currently available are unsatisfactory because, a) they do not determine 3-oxo-bile acids which are present in fecal extracts, b) oxo-bile acids are partially destroyed during alkaline hydrolysis, and c) acidic fecal pigments interfere with spectrophotometry at 340 nm. We are developing a new method which will overcome these difficulties. Oxo- acids will be reduced with sodium borohydride before alkaline hydrolysis, effectively protecting them from destruction and making possible 3-oxo-bile acid determination. A new indirect fluorometric method of assay will circumvent the pigment problem and make time-consuming purification of crude fecal extracts unnecessary. In this method, 3alpha- and 3-beta-hydroxy-bile acids reduced NAD to NADH in a reaction catalyzed by hydroxy-steroid oxidoreductase. NADH then reduces nonfluorescent resazurin to fluorescent resorufin in a reaction catalyzed by diaphorase. Fluorescence is excited at 565 nm and read at 580 nm wavelengths at which fecal pigment absorption is minimal. Systems for fecal bile acid extraction and enzymatic conjugated bile acid hydrolysis are being studied in an effort to reduce analysis time. A system for quantitative analysis of individual fecal bile acids or groups of fecal bile acids separated by thin-layer chromatography, which can be used with the assay, is also being developed.